Hepatocellular carcinoma (HCC) is a deadly cancer that affects more Veterans than the general American population. Risk factors include hepatitis virus infection, obesity, aging, alcohol/drug abuses, toxic exposure, and genetic/epigenetic predisposition, which collectively promote genetic mutations, cell proliferation and signaling pathway dysfunctions, leading to cancer. Significantly, men are affected 3 to 6 times higher than women in HCC. The male predominance in HCC has been a long-standing enigma in the medical field. The male sex hormone androgen and its receptor, androgen receptor (AR), exacerbate the oncogenic processes, including promotion of hepatitis viral replication, activation of oncogenes and signaling pathways, and repressing tumor suppressor activities, thereby exerting male preference in liver cancer. Hence, there are significant interests in antiandrogens as therapeutics in treatments of HCC in the clinical field. Importantly, we have detected the expression of constitutively active AR variants, e.g. AR-V7, in selected HCC specimens. Since these AR variants play important roles in metastatic advances in prostate cancer, their detection in HCC suggests that they could also exert oncogenic functions in liver cancer. Furthermore, we have identified a male-specific positive feedback loop, in which a male-specific oncogene TSPY interacts and amplifies AR and AR-V7 transactivation of target genes, including its own gene, in ligand-dependent and independent manners respectively, thereby potentially affecting the effectiveness of antiandrogen therapeutics for HCC patients. TSPY is the gene for the gonadoblastoma locus (GBY) on the Y chromosome. It is a cell cycle regulator, dysfunction of which promotes cell proliferation and oncogenesis. TSPY-positive HCC patients have worse survival rate than the negative ones while nuclear locations of AR/AR-V7 signify poor prognosis. Hence, understanding the mechanisms of actions of this male- specific positive feedback loop between AR/AR-V7 and the Y-located TSPY gene in hepatocarcinogenesis will provide the scientific foundation for translational applications in diagnosis, prognosis and clinical trials of antiandrogens as therapeutics in treatments of HCC. The project focuses on the synergistic and oncogenic functions of the male-specific positive feedback loop of AR/AR-V7 and TSPY in hepatocarcinogenesis under 3 specific aims. First, the expression patterns of TSPY, AR and AR-V7 in paired HCC tumor/non-tumor RNA samples and pathological specimens will be analyzed to substantiate the existence of such male-specific positive feedback loop and to evaluate if their expression patterns, including cytological locations, can be used as diagnostic and prognostic signatures for the patients. Second, the mechanisms of AR and AR-V7 transcriptional activation of the Y-located TSPY will be studied in HCC cells and mouse livers using hydrodynamic transfection strategy. Third, the oncogenic functions of TSPY, AR, and AR-V7 in HCC will be studied by stable hydrodynamic transfection to the livers of whole mice. Their ability to induce hepatic oncogenesis will be evaluated under normal conditions and tumorigenic predisposition. The global views on the mechanisms of their synergistic actions in hepatocarcinogenesis will be characterized with transcriptome and cistrome analyses in terms of differential gene expression, target gene preferences, and dysregulation of oncogenes, tumor suppressors and signaling pathways associated with cell growth, proliferation and tumorigenesis. We will use the resulting animal models to evaluate antiandrogen drugs as therapeutics in HCC treatments. The proposed research will provide critical insights on the molecular mechanisms of the male sex hormone receptor AR/AR-V7 and synergistic actions with the Y chromosome-located oncogene TSPY in the male-specific positive feedback loop in hepatocarcinogenesis. Success of the project will provide the scientific basis for translational applications in development of AR/AR-V7 and TSPY-based diagnosis, prognosis and therapeutic strategies in clinical management of HCC; and will greatly benefit the health care delivery to the Veterans predisposed to or suffering from liver cancer.